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  • Positiv "Negativ control" using NEXTflex Small RNA Kit

    Hi,

    I hope someone can answer my questions and bring some light into my darkness.

    I am using the NEXTflex Small RNA Kit in CLIP experiments to analyze mRNA binding motifs. So I immunoprecipitate RNP, do a proteinase K treatment, and precipitate previously bound RNA by TRI reagent. It work well so far and after using that Kit I got amplified products between 140 and 180 nts.

    So far, so good.

    Yesterday I though I might need a positiv and a negative control, respectively ... just checking! So I used the microRNA control (part of the kit) as positive control and added just water as negativ control (for ligations steps). After the second ligation I gel-purified my products by using a 12% UreaPA gel ... mainly to get rid of the adapterdimer. Unfortunately, I obtain a "postive band" in both samples after PCR, means for the 21 nt microRNA and the negative control "water".

    How can that be!? My problem is that I already got some products for sequencing, but now I am not sure if I have real data or just this "postive negative control" thing. Is someone able to advice me somehow!? That would be endless generous

    Thanks a lot.
    Best, Carlo

  • #2
    Hi Carlo,

    The positive control miRNA should give you a band size of approximately 146nt. The adapter dimer should be 120 nt. If you email me your positive and negative control gel images to: [email protected] we should be able to help you.

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